人端粒酶亲和纯化方法的建立及其初步鉴定

Establishment and pilot identification of affinity purification for human telomerase

目的：建立一种亲和纯化人端粒酶的手段。方法：酶切鉴定人端粒酶RNA（hTR）质粒，所切下的hTRcDNA片段用生物素标记后与链霉亲和秦琼脂糖珠耦连，用高端粒酶活性的胃肿瘤细胞提取液与之反应，利用端粒酶核糖核蛋白的特性，形成链霉亲和秦琼脂糖珠-生物素标记hTRcDNA－hTR-端粒酶蛋白组份复合物，核酸解离液洗脱后获得亲和纯人端粒酶。其活性用宝灵曼公司端粒酶试剂盒检测，定量利用BeckmanDU－640检测仪进行。结果：酶切鉴定的结果与hTR质粒图谱吻合。生物素hTRcDNA与链霉亲和素琼脂精珠耦连量为31．71μg，耦连率为98．38％。纯化后的端粒酶活性保持良好，相对纯化程度为108．46。结论：本研究中建立的方法是一种可行的端粒酶纯化手段，可用于人端粒酶的深入研究。

Objective: To establish a method for affinity purification of human telomerase- Methods: A fragment of human telomwrase RNA (hTRcDNA) was purified and collected with electroelution after the fragment was identified with testriction enzyme digestion- The affinity chromatography beads were prepared with binding of biotinylated hTRcDNA fragment with streptavidin agarose beads- The purification was based on the ribonucleoprotein property of teIomerase with hTRcDNA cornplementary to hTR template component.After binding reaction between the cancer cell extract with high telomerase activity and the affinity chromatography beads was set up, a complex of the affinity chromatography bead-telomerase was formed. Bound telomerase was eluted with nucleic acid denaturation reagent. TeIomerase activity was detected with telomerase PCR-ELISA. The quantity of protein and nucleic acid was assayed with Beckman DU-64o spectrophotometer. ResuIts: The result of restriction fragment analysis tallied with the structural map of hTR plasmid vector. The binding quantity and the rate of biotinylated hTRcDNA with streptavidin agarose beads were 31.71 pg and 98. 38% respectively. The affinity-purified telomerase kept active and its relative purity was lO8.46. Conclusion: Our purification method provides a feasible tool for further studies of human telomerase.